Intact microbial agents are known to have immunomodulatory activity as demonstrated by anti-infectious activity, anti-cancer activity, and adjuvant activity. This activity is evidenced by an increase in both humoral and cellular immune response. The active components of these microbial agents, as found, for example, in microbial agents of the class mycobacteriaceae, nocardia, and micrococcus, consist of the peptidoglycan cell wall skeleton and more particularly the repeating N-acetylglucosaminyl-N-acetylmuramyl peptide units. From this peptidoglycan, N-acetylmuramyl-L-alanine-D-isoglutamine, also known as muramyl dipeptide (MDP), has been identified as the minimal unit possessing immunological activities.
A large number of MDP derivatives have been synthesized and shown to possess immunological activity. While the monosaccharide muramyl dipeptides are immunilogically active, studies have established that the disaccharide dipeptide, which corresponds to the basic repeating unit of the cell wall peptidoglycan, possesses greater immunomodulating activity. This is shown, for example, by the anti-cancer activity of the monomeric units when given intravenously to mice bearing the Lewis lung carcinoma or the MCA mammary carcinoma (see, e.g., Sava et al., Cancer Immunology Immunotherapy, 15, 84-86 (1983)). Similarly, in humans, immunotherapy utilizing a disaccharide peptide derivative, such as N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmit oyl, has been more effective than a related monosaccharide peptide derivative, such as N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-phosphatidylethanolamine (see, e.g., Vosika et al., Journal of Immunotherapy, 10, 256-266 (1991)). Disaccharide peptides have also demonstrated enhanced efficacy in vaccine preparations.
The general use of the disaccharide peptide and related analogues has, however, been hampered by the cost of production. Typically, either the disaccharide dipeptide or the basic disaccharide itself (N-acetylglucosaminyl-N-acetylmuramic acid) is isolated from the cell walls of micrococcus lysodysticus through a process of cell fractionation, delipidation, enzymatic digestion, and extensive column chromatography. Alternative methods for isolation of the disaccharide and/or disaccharide dipeptide from biomass have included purification from the peptidoglycan complex excreted in penicillin treated cultures of a Brevibacterium Diverticulum mutant, enzymatic preparation from peptidoglycan of Actinomadura R39, and enzymatic preparation from hydrolysate of L. plantanum cell wall. The yields from such biologically based procedures are typically extremely low, on the order of 2% or less.
A variety of routes for the preparation of disaccharide structures by chemical synthesis have been reported as well. While a number of the reported routes have been successful in producing a disaccharide, the chemical synthetic methods have typically been characterized by low yields, large numbers of steps, difficult to handle reagents, stringent requirements on reaction conditions and/or lack of flexibility with regard to starting materials and products.
For example, methods of preparing disaccharides based on the condensation of a N-protected glucosaminyl donor with the hydroxyl group of glycosyl acceptor have been disclosed. The reported methods include condensations of N-protected glucosaminyl donors having a variety of leaving and/or activating groups (e.g., bromo, chloro, fluoro, methylthio, phenylthio, oxazoline, and trichloroacetimidate) with a glycosyl acceptor. Reports of condensations employing an N-protected 4-hydroxymuramic acid ester derivative as the acceptor have been extremely rare, however, perhaps because of the increased steric hindrance provided by the lactyl ether group. In order to avoid this problem, synthetic routes to protected glucosaminyl muramic acid ester compounds have typically delayed the introduction of the lactyl group until after the glycosylation step.
The condensation of an N-protected glucosaminyl chloride donor with an N-protected 4-hydroxymuramic acid ester acceptor has been disclosed. The condensation, however, is carried out in the presence of at least a full equivalent of silver triflate and must be run under a very specific set of reaction conditions which include the absence of base.
There is, accordingly, a continuing need for improved processes for the production of disaccharide dipeptide compounds via a chemical synthesis which includes the direct glycosylation of an N-protected 4-hydroxymuramic acid ester derivative.